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Mouse Embryonic Diastema Region Is an Ideal Site for the

Development of Ectopically Transplanted Tooth Germ
Yiqiang Song,1† Mingquan Yan,1 Ken Muneoka,1 and YiPing Chen1,2*
1
Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana
2
Department of Oral Biology, Ohio State University Health Sciences Center, Columbus, Ohio
*
Correspondence to: YiPing Chen, Department of Oral Biology, Ohio State University College of Dentistry, 305
W. 12th Avenue, Columbus, OH 43210. Email: chen.1278@osu.edu
†
Dr. Song’s present address is Department of Oral Biology, University of Illinois at Chicago College of
Dentistry, 801 S. Paulina Street, Chicago, IL 60612.
The publisher's final edited version of this article is available free at Dev Dyn
See other articles in PMC that cite the published article.

  Other Sections▼
o Abstract
o INTRODUCTION
o RESULTS AND DISCUSSION
o EXPERIMENTAL PROCEDURES
o References

Abstract
The anterior eye chamber and the kidney capsule of the mouse have been traditionally used
for long-term culture of tooth germ grafts. However, although these sites provide an
excellent growth environment, they do not represent real in situ sites for the development of
a grafted tooth germ. Here, we describe a protocol to transplant a tooth germ into the
mandibular diastema region of mouse embryos using exo utero surgery. Our results
demonstrate that the mouse embryonic diastema region represents a normal physiological
environment for the development of transplanted tooth germs. Transplanted tooth germs
developed synchronically with and became indistinguishable from the endogenous ones.
These ectopic teeth were vascularized and surrounded with nerve fibers, and were able to
erupt normally. Thus, the exo utero transplantation approach will provide a new avenue to
study tooth development and regeneration.
Keywords: tooth germ, tooth development, exo utero surgery, diastema
  Other Sections▼
o Abstract
o INTRODUCTION
o RESULTS AND DISCUSSION
o EXPERIMENTAL PROCEDURES
o References

INTRODUCTION
Given the relatively concise morphology and the ease with which it can be accessed,
manipulated, cultured, and grafted ex vivo, the mouse tooth germ has long been an
excellent model to study vertebrate organogenesis (Thesleff et al., 1996). Several
approaches have been developed to study tooth development ex vivo. The in vitro organ
culture has been routinely used, in which tooth germs are cultured in the Trowell type organ
culture dish (Thesleff et al., 1996). This method provides an easy and quick culture for
short-term growth of tooth germ and also provides an openly accessible environment for
the manipulation of tooth germ such as addition of growth factors. However, the artificial
culture condition limits not only the length of culture time but also the shape and size of
explanted tooth germs. The other method that has been used extensively for long-term tooth
germ culture is the kidney capsule culture in which tooth germs or organ primordial tissue
are grafted and cultured underneath the kidney capsule of adult mice (Morio, 1985).
Grafted tooth germs can develop into a well-differentiated tooth (Zhang et al., 2003b).
Actually, many parts of mouse body have been tested for hosting grafted tooth germs.
These sites include the anterior eye chamber (Kollar and Fisher, 1980; Yoshikawa and
Kollar, 1981; Lumsden and Buchanan, 1986), renal subcapsular site (Bartlett and Reade,
1973), subcutaneous tissue (Ivanyi, 1965), cheek pouch (Al-Talabani and Smith, 1979), and
spleen (Ishizeki et al., 1987). In addition, chick embryonic wing buds were also shown to
be an effective in vivo culture system for tooth development (Koyama et al., 2002).
However, even though some of these models provide an excellent growth environment for
tooth grafts, they represent a developmental and physiological environment that is distinct
from the oral cavity where tooth development normally occurs.
 The best and ideal site for ectopic tooth growth is within the oral cavity. The oral cavity of
the adult mouse has been successfully used to support the development of grafted tooth
germ (Ohazama et al., 2004; Nakao et al., 2007). However, the mouse after birth is full of
activity like daily food intake, which may influence and exert mechanical pressure on the
normal development of grafted tooth germ. It would be a better choice to nurture a tooth
germ in the oral cavity of the mouse at a comparable developmental stage. In mice, a
toothless region called diastema exists between the incisor and the first molar. This region
is believed to be filled with incisor, canine, and premolars before their disappearance
during the rodent evolution (Luckett, 1985; Peterkova et al., 1995). Today, the rudimentary
tooth germs still can be observed in the upper diastema of the mouse or sibling vole
embryos, and then these tooth germs are removed apoptotically through an unknown
mechanism (Keranen et al., 1999). On the other hand, the initiation of tooth development in
the mandibular diastema region is evidenced by a weak expression of Shh just anterior to
the first molar primordia (Mustonen et al., 2004). Accordingly, disturbance of the normal
gene regulation network in this area may results in an extra tooth (Mustonen et al., 2003;
Zhang et al., 2003a; Tucker et al., 2004; Kassai et al., 2005). Based on these observations,
we reasoned that the mandibular diastema region could be an ideal site for the development
of a tooth germ in addition to the endogenous tooth forming loci. To test this hypothesis,
we carried out an exo utero surgery to transplant tooth germ. Here, we describe this method
and demonstrate that transplanted tooth germs develop normally in the mouse embryonic
diastema region, indistinguishable from the endogenous ones.

  Other Sections▼
o Abstract
o INTRODUCTION
o RESULTS AND DISCUSSION
o EXPERIMENTAL PROCEDURES
o References

RESULTS AND DISCUSSION

Transplantation of Tooth Germ to the Mandibular Diastema of Mouse Embryo
by Exo Utero Surgery
One of the greatest advantages of using avian embryo as a developmental biology model is
the accessibility of its embryos throughout the entire developmental process. In the mouse,
however, accessing to the developing embryo is always difficult and poses a great
challenge due to the complex architecture of the mouse gestation organ. The finding that
mouse embryos can develop normally outside the uterine myometrium has substantially
extended the accessibility of mouse embryo to a much earlier stage, and moreover, such an
exo utero mouse embryo can survive some surgical manipulations to term (Muneoka et al.,
1986a,b). Benefiting from these findings, exo utero surgery has evolved to become a
powerful tool to study mouse embryonic development (Ngo-Muller and Muneoka, 2000). It
has been widely used in studies of nerve system development such as migration of the
neural crest cells during early mouse embryogenesis (Serbedzija et al., 1992), limb
development and regeneration (Han et al., 2003), as well as in teratology research where
effects of chemicals on the developmental mouse embryos can be studied. A recent review
has summarized the applications of mouse exo utero development system in biological
studies (Hatta et al., 2004).
We were originally seeking to identify an ideal site for the best development and growth of
ectopically grafted tooth germ or recombinant tooth germ after genetic modification in vitro
(Song et al., 2006). Compared with the kidney capsule, the anterior eye chamber, or other
ectopic loci, the oral cavity of the mouse appears to represent the most natural environment
for tooth development. The diastema, a toothless region between the incisor and molar, has
been demonstrated to host a full array of teeth before they were evolutionally lost. Teeth
indeed develop in the mandibular diastema of some genetically modified mice (Mustonen
et al., 2003; Tucker et al., 2004; Zhang et al., 2003a; Kassai et al., 2005). We, therefore,
deduced that the mandibular diastema of the mouse embryo had an intrinsic ability to
support tooth development.
Although exo utero surgery can be carried out on mouse embryos as early as embryonic
day (E) 11.5, a better survival rate is achieved with surgery on E13.5 or older embryos
(Muneoka et al., 1986a). On the other hand, tooth germs of E13.5 mouse embryos are at the
bud stage, which provides an ideal size and solidity for surgical operation compared with
that from earlier stages. Thus, we began with transplanting E13.5 mouse tooth germs into
the mandibular diastema of E13.5 host embryos. Such an arrangement allows the
endogenous tooth primordia to be the control, which provides standard developmental
indexes side by side for the evaluation of the ectopic tooth growth. Here, we further took
advantage of tooth germs from Rosa26-Egfp mouse embryos, of which tissue can be easily
detected and distinguished from the host embryo under the fluorescent microscope by
activation through fluorescein isothiocyanate (FITC) -filter set.
For exo utero surgery, the incisor tooth germ was isolated from the E13.5 Rosa26-Gfp
mouse embryo. At this stage, the incisor primordia is much better for transplantation
experiments due to their smaller size compared with the molar tooth germs. It can be easily
grafted into a small incision made in the diastema region of host embryos. As described in
detail in the Experimental Procedures section, the same stage embryos were properly
exposed from the uterus of an anesthetized female mouse. As shown in Figure 1A, an E13.5
host embryo was exposed after opening the uterus and removing the extraembryonic
membrane in a timed-pregnant mouse. Under adequate illumination, details of the embryo
can be easily defined under the dissection microscope. Before grafting into the small
incision made in the diastema region, the isolated incisor germ was carried on a thin copper
wire to the embryonic mandible. The tooth germ was then carefully placed into the incision
with the epithelial side face up. By piercing the neighboring host tissue with the copper
wire, the transplanted tooth germ was fixed at the incision site. Figure 1B shows a
transplanted Rosa26-Egfp incisor primordia fixed at the mandibular diastema region of an
E13.5 wild-type host embryo. Under the activation light through the FITC-filter set, the
ectopic tooth germ was emanating green fluorescent light.

Fig. 1
Ex utero surgery for tooth germ transplantation into the mandibular diastema of
embryonic day (E) 13.5 mouse embryo. A: An E13.5 mouse embryo is exposed after
opening of the uterus and extraembryonic membrane. B: An E13.5 host embryo
receives an incisor (more ...)
Fig. 1
Ex utero surgery for tooth germ transplantation into the mandibular diastema of embryonic day (E) 13.5
mouse embryo. A: An E13.5 mouse embryo is exposed after opening of the uterus and extraembryonic
membrane. B: An E13.5 host embryo receives an incisor tooth germ from E13.5 Rosa26-Egfp embryo in
the mandibular diastema region. The grafted tooth germ that was fixed by two copper wire pins showed
fluorescence under the fluorescent microscope. C: A full-term host mouse carrying the grafted tooth
germ, as indicated by fluorescence. AM, amnion membrane; CB, cotton ball; CW, copper wire pin; E,
eye; FL, front limb; GT, GFP mouse embryo derived tooth germ; H, head; MD, mandible; MX,
maxillary.

Transplanted Tooth Germ Develops Normally in the Diastema

Histological analysis of a full-term host embryo shows a well-developed tooth germ in the
transplanted site (Fig. 2A). Both epithelial and mesenchymal structures of the transplanted
incisor are comparable to that of the endogenous incisors (Fig. 2B). To confirm the
supportive role of host tissue in the ectopic tooth development, we further examined blood
vessel invasion and innervation of the transplanted tooth. By immunohistochemical staining
using antibody against laminin, a molecular marker for blood vessel endothelial cells, we
were able to detect blood vessels in the dental pulp of both the endogenous and the
transplanted teeth (Fig. 2C,D), clearly demonstrating the presence of blood vessels in the
transplanted tooth. The invasion of blood vessels provides the transplanted tooth with
nutrients through the blood supplies from the host. Moreover, using antibodies against
neurofilament, we have detected nervous termini around the transplanted tooth, indicating
that the transplanted tooth will have the similar potential as the endogenous teeth to be
innervated (Fig. 2E,F). Thus, the transplanted tooth germ develops synchronically with the
endogenous tooth in the host embryo. Based on the facts that blood vessels are not present
in the developing mouse tooth before the cap stage (E14.5; Luukko et al., 2005) and that
tooth innervation does not occur until birth (Tsuzuki and Kitamura, 1991; Kettunen et al.,
2005), we conclude that these blood vessel cells and neuron cells within or surrounding the
transplanted tooth are derived from the host.

Fig. 2
Development of transplanted tooth germ in the term host mouse. A: A
coronal section through a host mouse head shows a grafted incisor
germ (GI) in the mandibular diastema adjacent to the endogenous
incisor germ (EI). B: Higher magnification of A shows (more ...)

Fig. 2
Development of transplanted tooth germ in the term host mouse. A: A coronal section through a host
mouse head shows a grafted incisor germ (GI) in the mandibular diastema adjacent to the endogenous
incisor germ (EI). B: Higher magnification of A shows a well developed ectopic incisor (arrow),
comparable to the adjacent endogenous one. C,D: Innmunohistochemical staining shows cells positive
for laminin, a molecular marker for blood vessel endothelial cells, in the dental pulp of a transplanted
incisor in a host mouse at birth (C), compared with the endogenous incisor at the same age (D). E,F:
Immunostaining for neurofilament shows nerve termini (arrow) around a transplanted incisor (E), and in
the endogenous control tooth (F). AB, alveolar bone; AM, ameloblast; BV, blood vessel; D, dentin; DP,
dental pulp; EI, endogenous incisor; GI, grafted incisor; OB, odontoblast; T, tongue; N, nerve.
Of interest, when E13.5 embryos were used as hosts for tooth transplantation, all the hosts
(21 in total) that were monitored died within 1 day after caesarian section. Examination of
gross morphology identified the cleft palate phenotype in all the surgical embryos,
explaining the postnatal lethality. It is known that the palate shelves elevated from a
perpendicular to the horizon position and fused to each other above the tongue between
E13.5 and E14.5. Therefore, our surgical operation in the oral cavity of E13.5 embryo must
have disturbed such an elevation and/or fusion process of the palate shelves, resulting in the
cleft palate formation.
Because the palate shelves have already fused in E14.5 mouse embryos, we decided to use
E14.5 mouse embryos as host for tooth transplantation, in hope of getting living pups with
an ectopic tooth in their mouth. As expected, none of the pups (25 in total) that received
tooth germ transplantation at E14.5 developed a cleft palate phenotype. The mice were fed
by a foster mother immediately after C-section, and grew up as normal mice. Figure 3
shows such a weaned mouse carrying an ectopic incisor growth in the mandibular diastema.
These results clearly demonstrate that the transplanted tooth germ can develop normally in
the diastema region of mouse embryo and can eventually erupt and form as a normal tooth
in the host mouse oral cavity.

Fig. 3
Growth of a transplanted incisor in the mandibular diastema of a host
mouse. The grafted tooth erupted and grew at the comparable size as the
endogenous ones. LI, lower incisor; GI, grafted incisor; UI, upper
incisor; N, nasal

Fig. 3
 Growth of a transplanted incisor in the mandibular diastema of a host mouse. The grafted tooth erupted
and grew at the comparable size as the endogenous ones. LI, lower incisor; GI, grafted incisor; UI, upper
incisor; N, nasal

The mechanism of how some vertebrates lost their teeth during evolution remains elusive.
It could be a result of loss of gene function that is critical for tooth development. In avian,
the remnant of developing tooth primordia can be found in the oral cavity too, and the
abortion of tooth development is at least partially due to the loss of odontogenic Bmp4
expression (Chen et al., 2000). On the other hand, tooth agenesis could also be a result of
an active inhibitory mechanism that is present in the oral cavity to repress tooth
development. It is evidenced that Gas1, a diffusible protein acting as Shh antagonist, is able
to sequester Shh and, therefore, to block Shh function in the mouse diastema region. Such
an antagonistic mechanism may be used to delineate the border of tooth forming sites and
the diastema region at the beginning of tooth development, or required to constantly inhibit
tooth development within the diastema (Cobourne et al., 2004). Nevertheless, we show here
that, at or after E13.5, the mandibular diastema of mouse embryo serves as an excellent site
to support tooth development. Our results suggest that the grafted tooth germ is able to
override any inhibitory mechanism or can develop independent of the inhibitory
mechanism. Alternatively, the constant inhibitory mechanism discussed above may have
disappeared in the diastema region at the time when tooth germ is grafted.
Similar to many other ectopic sites in living animals, particularly the mouse kidney capsule,
the embryonic diastema region appears to be excellent for grafted tooth development and
differentiation. However, the approach we describe here also has several advantages, such
as (1) it makes it possible to study tooth eruption of embryonic lethal mutant mice, and (2)
it permits studies on tissue interactions between grafted tooth germ and the host supportive
tissues in the oral cavity. On the other hand, this approach has its limitation as well. The
size of a graft is a concern. For example, an E13.5 molar germ was proven too big to be
grafted properly. In conclusion, while the diastema region of the mouse mandible does not
grow endogenous teeth, it does fully support tooth formation when ectopic tooth germ is
grafted.

  Other Sections▼
o Abstract
o INTRODUCTION
o RESULTS AND DISCUSSION
o EXPERIMENTAL PROCEDURES
o References

EXPERIMENTAL PROCEDURES
Tooth Germ Preparation and Transplantation by Means of Exo Utero Surgery
Tooth germs at the bud stage were used as donors in this study. Embryos at E13.5 were
collected from timed pregnant CD-1 or Rosa26-Egfp mice. The incisor tooth germs were
isolated from the mandibles and kept in ice-cold PBS for use. A fine copper pin was used to
pierce through each tooth germ at the mesenchyme, making it easier to transfer the tooth
germ from the dish to the surgical embryo with forceps. Exo utero surgery was performed
as described previously (Ngo-Muller and Muneoka, 2000). In this study, we used E13.5 and
E14.5 embryos as recipients for tissue transplantation. Briefly, timed pregnant CD-1 mice
at 13.5 or 14.5 gestation were anesthetized by intraperitoneal injection of Nembutal at a
dose of 10 μl/g body weight. After properly anesthetized, the mouse was placed on a
surgical platform. The skin and body wall of the abdomen were sequentially opened by
scissors and forceps. The uterus was subsequently opened with iridectomy scissors at the
opposite of placental side in a continuous longitudinal incision. By rolling a dry cotton-
tipped applicator between the placenta and the uterus, the unwanted embryos were removed
with minimal bleeding from the uterus. Usually, four embryos were left in the abdomen of
each female for grafting. Blood and debris were washed away with lactated Ringer’s
solution before further operation. Extra care needs to be taken to avoid damaging the major
blood vessels in the extraembryonic membrane when an incision was made in the
membrane. The head of the host embryo was allowed to pop out through the incision on the
extraembryonic membrane, and then was properly positioned with the help of sterile cotton
balls as cushions. A small slit was made by fine forceps within the diastema region of the
host’s mandible. Donor tooth germ was inserted into the incision with the dental epithelial
side facing the oral cavity, and then was fixed by fine copper pins. After completion of
tissue transplantation, the extraembryonic membrane was closed by suturing. The abdomen
cavity was then sutured after cotton balls were removed and the abdomen cavity was totally
cleaned with lactated Ringer’s solution. Typically, the mouse wakes up within 1 hr after
surgery. A warm heating pad was used to help the mouse maintain body temperature during
this process.
The pregnant female mouse was housed and closely monitored in an isolated cage until
term. To collect experimental embryos, the host female mouse was killed, and surgical pups
were collected through C-section. For further development purposes, the operated embryos
were stimulated to begin breathing by rubbing cotton-tipped applicator on their tail tips.
Any fluids in their mouth were also cleaned with a cotton-tipped applicator. When they
were breathing regularly and moving about, the experimental newborns were transferred to
the cage with a nursing mother that had delivered her litter at the same day or 1 day before.
An animal protocol for the exo utero surgery was approved by the IACUC of Tulane
University. The entire surgery procedure strictly followed the animal protocol.
Histology and Azan Dichromic Staining
The recipient mice were killed at birth. The heads were harvested and fixed in 4%
paraformaldehyde (PFA)/ PBS, de-calcified by demineralization in 0.1 M
ethylenediaminetetraacetic acid/PBS for a week with several changes of solution. Samples
were then dehydrated through graded ethanol, cleared with xylene, embedded in paraffin,
and sectioned at 10 μm. Sections were processed with Azan di-chromic staining, as
described previously (Presnell and Schreibman, 1997).

Immunohistochemistry
The recipient mice were collected at the delivery day, and the heads were removed and
processed for frozen sections. OCT-embedded samples were sectioned at 10 μm, and the
cryostat sections were fixed for 30 min at room temperature with 4% PFA and analyzed by
indirect immunohistochemical staining. The rabbit anti-mouse laminin antibody
(Chemicon, Temecula, CA) was used as the primary antibody specific for blood vessel
endothelial cells, and the rabbit anti-mouse neurofilament 200 antibody (Sigma, St. Louis,
MO) was used as the primary antibody for nerve cells. The horseradish peroxidase-coupled
goat anti-rabbit IgG were used as the secondary antibody (Sigma). Immunohistochemical
staining was performed according to the protocol provided with the antibodies by the
manufacturers.

Acknowledgments
Y.P.C. and K.M. were supported by the NIH.
 Grant sponsor: the NIH; Grant number: DE15123; Grant number: DE16623; Grant number:
HD43277.

  Other Sections▼
o Abstract
o INTRODUCTION
o RESULTS AND DISCUSSION
o EXPERIMENTAL PROCEDURES
o References

References

1. Al-Talabani NG, Smith CJ. A histological study of the effect of developmental stage of tooth-germ on
transplantation to hamster cheek pouch. Arch Oral Biol. 1979;24:933–937. [PubMed]

2. Bartlett PF, Reade PC. The antigenicity of mouse tooth germs. I. Isogenic and allogenic transplantation.
Transplantation. 1973;16:479–488. [PubMed]

3. Chen YP, Zhang Y, Jiang TX, Barlow AJ, St Amand TR, Hu Y, Heaney S, Francis-West P, Chuong CM,
Maas R. Conservation of early odontogenic signaling pathways in Aves. Proc Natl Acad Sci U S A.
2000;97:10044–10049. [PMC free article] [PubMed]

4. Cobourne MT, Miletich I, Sharpe PT. Restriction of sonic hedgehog signalling during early tooth
development. Development. 2004;131:2875–2885. [PubMed]

5. Han MJ, Yang XD, Farrington JE, Muneoka K. Digit regeneration is regulated by Msx1 and Bmp4 in fetal
mice. Development. 2003;130:5123–5132. [PubMed]

Stem/Progenitor Cell–Mediated De Novo Regeneration of Dental

Pulp with Newly Deposited Continuous Layer of Dentin in an In
Vivo Model
George T.-J. Huang, D.D.S., M.S.D., D.Sc., 1,2,3 Takayoshi Yamaza, D.D.S., Ph.D.,4,*
Lonnie D. Shea, Ph.D.,5 Farida Djouad, Ph.D.,3 Nastaran Z. Kuhn, Ph.D.,3 Rocky S. Tuan,
Ph.D.,3,† and Songtao Shi, D.D.S., Ph.D.4
1
Section of Oral and Diagnostic Sciences, Division of Endodontics, College of Dental Medicine, Columbia University,
New York, New York.
2
Department of Endodontics, Prosthodontics, and Operative Dentistry, College of Dental Surgery, Dental School,
University of Maryland, Baltimore, Maryland.
3
Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases,
National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland.
4
Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, Los Angeles,
California.
 5
Department of Chemical and Biological Engineering, McCormick School of Engineering, Northwestern University,
Evanston, Illinois.
Corresponding author.
Address correspondence to: George T.-J. Huang, D.D.S., M.S.D., D.Sc., Department of Endodontics, Boston University,
Henry M. Goldman School of Dental Medicine, 100 E. Newton St., Boston, MA 02118. E-mail:Email: gtjhuang@bu.edu
*
Present address: Department of Oral Anatomy and Cell Biology, Kyushu University Graduate School of Dental Science,
Fukuoka, Japan.
†
Present address: Department of Orthopaedic Surgery and Center for Celluar and Molecular Engineering, University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Received July 25, 2009; Accepted September 8, 2009.

This article has been cited by other articles in PMC.

  Other Sections▼
o Abstract
o Introduction
o Materials and Methods
o Results
o Discussion
o References

Abstract
The ultimate goal of this study is to regenerate lost dental pulp and dentin via
stem/progenitor cell–based approaches and tissue engineering technologies. In this study,
we tested the possibility of regenerating vascularized human dental pulp in emptied root
canal space and producing new dentin on existing dentinal walls using a stem/progenitor
cell–mediated approach with a human root fragment and an immunocompromised mouse
model. Stem/progenitor cells from apical papilla and dental pulp stem cells were isolated,
characterized, seeded onto synthetic scaffolds consisting of poly-D,L-lactide/glycolide,
inserted into the tooth fragments, and transplanted into mice. Our results showed that the
root canal space was filled entirely by a pulp-like tissue with well-established vascularity.
In addition, a continuous layer of dentin-like tissue was deposited onto the canal dentinal
wall. This dentin-like structure appeared to be produced by a layer of newly formed
odontoblast-like cells expressing dentin sialophosphoprotein, bone sialoprotein, alkaline
phosphatase, and CD105. The cells in regenerated pulp-like tissue reacted positively to
anti-human mitochondria antibodies, indicating their human origin. This study provides the
first evidence showing that pulp-like tissue can be regenerated de novo in emptied root
canal space by stem cells from apical papilla and dental pulp stem cells that give rise to
odontoblast-like cells producing dentin-like tissue on existing dentinal walls.
  Other Sections▼
o Abstract
o Introduction
o Materials and Methods
o Results
o Discussion
o References

Introduction
REGENERATION OF dental pulp/dentin tissues in the pulp space of teeth serves the ultimate goal
of preserving teeth via endodontic approaches. Attempts to induce tissue regeneration in the
pulp space have been a longstanding quest. Pulp tissue regeneration has been explored
using the biodegradable synthetic material polyglycolic acid seeded with pulp cells, and
results showed pulp-like tissue formation in both in vitro and in vivo models.1–3 These
previous approaches were the proof-of-principle studies that only tested the formation of a
pulp-like soft tissue without dentin. From a clinical perspective, the following issues must
be considered when attempting to regenerate functional pulp/dentin tissues in a root canal
space: (i) regenerated pulp tissue must be vascularized although the blood supply is only
available from the apical end; (ii) newly differentiated odontoblasts should form on the
existing dentinal wall in the root canal space, and (iii) new dentin should be produced by
the new odontoblasts onto the existing dentin.4,5
Using a tooth slice model (horizontal section, 1 mm thick), it was shown that the stem cells
from human exfoliated deciduous teeth seeded onto the synthetic scaffolds that were fabri-
cated in the pulp chamber space formed well-vascularized pulp-like tissue in an in vivo
study model. In addition, odontoblast-like cells derived from the pulp-like tissue were
localized against the existing dentin surface.6 Tooth slice model was used to ensure blood
supply to the stem cell–seeded scaffolds. To date, there is a lack of evidence demonstrating
that the human pulp tissue can be regenerated in an emptied root canal space with only one
opening to the blood supply, and showing that the regenerated pulp tissue would form a
continuous layer of newly deposited dentin onto the existing dentinal walls. Using human
DPSCs from permanent teeth seeded onto a dentin surface, small amounts of discontinuous
dentin-like mineralized tissue on the existing dentin surface have been observed in vivo.7
Previously, we reported that the developing organ at the apex of the tooth, the apical
papilla, contained multipotent stem cells, named stem cells from apical papilla (SCAP), that
 were able to form pieces of pulp-dentin complex in an animal model.8,9 Moreover, SCAP
are cells from a developing tissue and are more robust than DPSCs in terms of population
doubling capacity, proliferation rate, telomerase activity, and cell migration ability. 8 We
hypothesized that vascularized pulp-like and dentin-like tissues can be regenerated in an
emptied root canal space with DPSCs and SCAP. Using a model system that includes ex
vivo–expanded human DPSCs and SCAP, synthetic scaffolds, human root segments, and
immunocompromised mice, we show here for the first time the regeneration of vascularized
pulp-like tissue and the formation of dentin-like mineral structures depositing onto the
existing dentinal wall in the root canal space.

  Other Sections▼
o Abstract
o Introduction
o Materials and Methods
o Results
o Discussion
o References

Materials and Methods

Sample collection
Normal human-impacted third molars (n = 12) with or without immature roots were
collected from healthy consenting patients (eight donors aged 16–24 years) in the Dental
Clinics at the University of Maryland and the University of Southern California. Freshly
extracted teeth were stored in serum-free cell culture medium and transported to the
laboratory for processing. Bone marrow was obtained from the long bone of healthy
consenting patients (aged 20–63) undergoing orthopedic surgery at the Walter Reed Army
Medical Center. Sample collection conformed to approved protocols by the respective
Medical Institutional Review Boards and the National Institutes of Health.
Cell culture
Isolation of DPSCs and SCAP was described previously. 8–11 In brief, pulp tissue and apical
papilla were removed from teeth, minced, and digested in a solution of 3 mg/mL
collagenase type I and 4 mg/mL dispase (Sigma-Aldrich, St. Louis, MO) for 30–60 min at
37°C. The digested mixtures were passed through a 70-μm cell strainer (Falcon; BD
Labware, Franklin Lakes, NJ) to obtain single-cell suspensions. Cells were seeded onto six-
well plates and cultured with α-minimum essential medium (Gibco–Invitrogen, Carlsbad,
CA) supplemented with 15–20% fetal bovine serum (FBS; Gemini Bio-Products,
Woodland, CA), 2 mM L-glutamine, 100 μM L-ascorbic acid-2-phospate, 100 U/mL
penicillin-G, 100 μg/mL streptomycin, and 0.25 μg/mL fungizone (Gemini Bio-Products)
and maintained in 5% CO2 at 37°C. Colony formation units of fibroblastic cells were
normally observed within 1–2 weeks after cell seeding and were passaged at 1:3 ratio when
they reached ~80% confluence. Heterogeneous populations of DPSCs and SCAP were
frozen and stored in liquid nitrogen at passages 0–2. Cells were thawed and expanded for
experimentation at passage 3. Human bone marrow mesenchymal stem cells (BMMSCs)
were isolated and cultured as previously described11–13 and were cultured in expansion
medium containing Dulbecco's modified Eagle's medium and 10% preselected FBS.
Heterogeneous populations of isolated colony formation units of fibroblastic cells were
used for all the assays and tissue regeneration experiments.
Flow cytometry
Cell aliquots of SCAP and DPSCs (>2 × 105 cells) were washed and resuspended in
phosphate-buffered saline (PBS) + 0.1% FBS, containing saturating concentrations (1:100
dilution) of the following conjugated mouse IgG1,κ anti-human monoclonal antibodies (BD
Biosciences, San Jose, CA): CD14-PE, CD34-PE, CD45-FITC, CD73-PE, CD90-FITC,
and CD105-PE for 1 h at 4°C. Cell suspensions were washed twice and resuspended in
0.1% FBS/PBS for analysis on a flow cytometer (FACS Calibur; BD Biosciences) using
the CellQuest Pro™ software (BD Biosciences).
Multiple lineage differentiation
Odonto/osteogenic differentiation
Cells were seeded onto 48-well plates, grown to ~70% confluence, and incubated in the
differentiation medium containing 10 nM dexamethasone, 10 mM β-glycerophosphate, 50
μg/mL ascorbate phosphate, 10 nM 1, 25 dihydroxyvitamin D3, and 10% FBS for 5 weeks.
Cultures were fixed in 60% isopropanol, and mineralization of extracellular matrix stained
with 1% Alizarin Red S.
Adipogenic differentiation
Cells were seeded onto 12-well plates, grown to subconfluence, and incubated in the
adipogenic medium containing 1 μM dexamethasone, 1 μg/mL insulin, 0.5 mM 3-isobutyl-
1-methylxanthine, and 10% FBS for 6 weeks. Cells were fixed in 10% formalin for 60 min,
washed with 70% ethanol, and lipid droplets were stained with 2% (w/v) Oil Red O reagent
for 5 min and washed with water.
Neurogenic differentiation
Cells at subconfluence in chamber slides were incubated in the neurogenic induction
medium Neurobasal A (Gibco-Invitrogen) with B27 supplement (GIBCO-BRL), 20 ng/mL
epidermal growth factor (EGF) (BD Biosciences), and 40 ng/mL fibroblast growth factor
(FGF) (BD Biosciences). After 4 weeks, cells were analyzed by immunocytofluorescence
for the expression of the neural cell marker, βIII-tubulin. Isotype-matched antibodies were
used as negative controls. After fixation in 100% ice-cold methanol, cells were incubated in
blocking buffer (32.5 mM NaCl, 3.3 mM Na2HPO4, 0.76 mM KH2PO4, 1.9 mM NaN3, 0.1%
[w/v] bovine serum albumin, 0.2% [v/v] Triton-X 100, 0.05% [v/v] Tween 20, and 5% goat
serum) for 30 min followed by the addition of monoclonal mouse anti-human βIII-tubulin
antibodies (Promega, Madison, WI) for 1 h at room temperature. After washing, cultures
were incubated with anti-mouse Alexa Fluor 594 for 1 h at room temperature and the cell
nuclei stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen,
Carlsbad, CA). Images were analyzed under a fluorescence microscope.
Myogenic differentiation
Cells were seeded onto 12-well plates, grown to subconfluence, and induced in the
myogenic medium containing 0.1 μM dexamethasone, 50 μM hydrocortisone, and 5% horse
serum for 6 weeks. Cells were then harvested for RNA isolation using RNeasy Mini Kit
(Qiagen, Valenicia, CA). cDNA was synthesized with SuperScript reverse transcription
(RT)-polymerase chain reaction kit (Invitrogen) followed by polymerase chain reaction
using Taq DNA polymerase (Invitrogen) to detect the expression of myogenic genes. The
following genes were examined with specific primers: MyoD1 (forward, 5′-AAG CGA
CCT CTC TTG AGG TA-3′; reverse, 5′-GCG CCT TTA TTT TGA TCA CC-3′),
myogenin (forward, 5′-TAA GGT GTG TAA GAG GAA GTC G-3′; reverse, 5′-CCA CAG
ACA CAT CTT CCA CTG T-3′), myosin heavy polypeptide 1 (forward, 5′-TGT GAA TGC
CAA ATG TGC TT-3′; reverse, 5′- GTG GAG CTG GGT ATC CTT GA-3′), and GAPDH
(forward, 5′-CAA GGC TGA GAA CGG GAA GC-3′; reverse, 5′-AGG GGG CAG AGA
TGA TGA CC-3′).
Scaffold fabrication
A copolymer of poly-D,L-lactide and glycolide (PLG) (75:25 molar ratio) (Boehringer
Ingelheim, Ingelheim, Germany) was employed to create porous scaffolds using a gas
foaming/particulate leaching process, and cells were seeded onto the scaffolds according to
the previously described procedures.14 Briefly, porous polymer scaffolds were formed by
mixing PLG microspheres and salt crystals (diameter 250–425 μm), which were then
loaded into a cylindrical mold of 5 (diameter) × 2 (height) mm. The mixture was then
compressed at 1500 psi, yielding solid disks and foamed in a pressure vessel using CO 2 at
850 psi. Subsequent solvent leaching rendered the scaffolds porous with pore diameters of
250–425 μm.
In vitro analysis of dental stem cells grown in scaffolds
Each PLG scaffold disk was cut into small pieces of ~1.5–2.0 mm3 to allow maximal stem-
cell attachment. Cells (107/mL) were suspended in cell culture medium, and 5 μL of cells
per scaffold piece were loaded into the polymer scaffold for a 5-min incubation period. For
the in vitro studies, the cell-seeded PLG scaffolds were placed in wells of 12-well plates,
and the culture medium was changed every 2–3 days. At different time points, the cell-
seeded scaffolds were processed for standard histological and scanning electronic
microscopy (SEM) studies to observe their attachment and morphology. The SEM
procedures followed the protocol as described previously.10,15
Preparation of root fragments of human teeth
Radicular portions of freshly extracted human teeth were horizontally sectioned into
segments of ~6–7 mm in length using an Isomet saw (Buhler, Lake Bluff, IL). The root
canal space was enlarged to ~1–2.5 mm in diameter. One end of the canal was sealed with
mineral trioxide aggregate (MTA) cement ~1 mm into the canal space, leaving the depth of
the canal space ~5–6 mm (Fig. 1). Root fragments were soaked at room temperature in 17%
ethylenediamine tetraacetic acid for 10 min and 19% citric acid for 1 min to remove the
smear layer,10,15 followed by treatment with betadine for 30 min and 5.25% NaOCl for 10–
15 min for sterilization. Fragments were then rinsed in sterile PBS, soaked in PBS, and then
incubated at 37°C for 3–7 days to remove residual sterilization agents and to ensure that
there is no microbial contamination.

FIG. 1.
 SCID mouse subcutaneous study model for pulp/dentin regeneration. The canal
space of human tooth root fragments (~6–7 mm long) was enlarged to ~1–2.5 mm in
diameter. One end of the canal opening was sealed (more ...)

FIG. 1.
SCID mouse subcutaneous study model for pulp/dentin regeneration. The canal space of human tooth
root fragments (~6–7 mm long) was enlarged to ~1–2.5 mm in diameter. One end of the canal opening
was sealed with MTA cement. SCID, severe combined immunodeficient; MTA, mineral trioxide
aggregate.

Insertion of cells/scaffolds into root fragments and implantation into mouse

subcutaneous space
Cells (107/mL) were seeded onto the PLG scaffolds for 5 min as described previously for in
vitro analysis. Immediately, the cells/PLG were inserted into the canal space of each root
fragment and kept in the wells of 12-well plates with a minimal amount of cell culture
medium. The tooth constructs were then implanted into the subcutaneous space on the back
of 6- to 8-week-old female severe combined immunodeficient mice (NOD.CB17-Prkdc-
scid/J; Jackson Laboratory, Bar Harbor, ME). Mice were anesthetized, and the dermal
space was created by blunt lateral dissection from a single dorsal midline incision. Each
mouse received two tooth fragments, one on each side. The wounds were sutured to obtain
primary closure. In parallel, root fragments with empty canal space were implanted into
severe combined immunodeficient mice as controls. Three to 4 months after the
transplantation, the mice were euthanized and the tooth fragments removed for histological
analysis. All animal procedures followed a protocol approved by the Institutional Animal
Care and Use Committee at the University of Maryland.
Histological examination
Samples (normal teeth and resected transplants) were fixed in 4% phosphate-buffered
paraformaldehyde, decalcified in 10–15% ethylenediaminetetraacetic acid for 1–2 months,
 paraffin embedded, longitudinally sectioned, and stained with hemotoxylin and eosin for
histological analysis. Some of the sections were used for immunohistochemical analysis.
Immunohistochemistry
Deparaffinized sections were immersed in 3% H2O2/methanol for 15 min to quench
endogenous peroxidase activity and incubated with primary antibodies (1:200 to 1:500
dilution) overnight at 4°C. Primary antibodies used were as follows: alkaline phosphatase
(ALP; LF-47), dentin sialoprotein (DSP, LF-21), and bone sialoprotein (BSP; LF-120),
provided by Dr. Larry Fisher (National Institute of Dental and Craniofacial Research,
National Institutes of Health, Bethesda, MD). Antibodies to CD105 were from BD
Biosciences and to human mitochondria were from Chemicon (Temecula, CA). Isotype-
matched control antibodies were used under the same conditions as the primary antibodies.
For enzymatic immunohistochemical staining, Zymed SuperPicTure polymer detection kit
(Zymed–Invitrogen, Carlsbad, CA) was used according to the manufacturer's protocol.
Sections were counterstained with hematoxylin.

  Other Sections▼
o Abstract
o Introduction
o Materials and Methods
o Results
o Discussion
o References

Results
In vitro characterization of dental stem cells
Cell marker analysis
Human DPSCs and SCAP consisting heterogeneous populations of stem and progenitor
cells were reproducibly isolated using established protocols. 8–11 Cells at passage 3 were
stained with antibodies against a number of cell surface markers and analyzed by flow
cytometry. CD14, CD34, and CD45 were not expressed, whereas CD73, CD90, and CD105
were expressed moderately to highly on these cells (Table 1). The surface marker
expression profile suggests that these cells were of typical mesenchymal cell lineages
similar to what normally is found in BMMSCs.16,17
 Table 1.
Surface Epitope Phenotype of Dental Stem/Progenitor Cellsa

Multipotentiality of SCAP and DPSCs

To verify that isolated dental cells qualified as stem/progenitor cells, we examined their
odonto/osteogenic, adipogenic, neurogenic, and myogenic potential with BMMSCs
included for comparison. Extracellular matrix production and mineralization by SCAP and
DPSCs after odonto/osteogenic stimulation was comparable to or greater than those of
BMMSCs (Fig. 2). SCAP and DPSCs were much weaker in adipogenicity because fewer
cells developed intracellular lipid droplet accumulation and showed delayed adipogenesis
compared with BMMSCs. Of those SCAP and DPSCs that did accumulate lipid droplets,
they did not expand to the size of adipocyte-like cells that emerged from BMMSCs (Fig.
2B, E, H insets). Cells under neurogenic induction underwent morphological changes (not
shown). In general, BMMSCs exhibited spherical cell bodies with long and multiple
cellular extensions, whereas SCAP and DPSCs still possessed spindle-shaped fibroblastic
cell bodies with mainly long cellular processes. We found a subpopulation of these cells
(20–30%) expressing the neurogenic marker βIII-tubulin (Fig. 2C, F, I). Under myogenic
stimulation, BMMSCs did not show any signs of myotube formation. SCAP and DPSCs
had a slender morphology and became elongated, but without myotube formation.
Myogenic gene expression analysis revealed that BMMSCs did not express any of the three
genes examined (myoD1, myogenin, and myosin heavy polypeptide 1), while myoD1 was
detected in SCAP and DPSCs after stimulation (Fig. 2J).

FIG. 2.
Multiple lineage differentiation properties of SCAP and DPSCs compared with
BMMSCs. (A, D, G) Odonto/osteogenic induction and Alizarin Red staining of
matrix mineralization. Insets: uninduced control. All scale bars including insets: 200
μm. (more ...)

FIG. 2.
Multiple lineage differentiation properties of SCAP and DPSCs compared with BMMSCs. (A, D, G)
Odonto/osteogenic induction and Alizarin Red staining of matrix mineralization. Insets: uninduced
control. All scale bars including insets: 200 μm. (B, E, H) Adipogenic induction and Oil Red O staining
of the accumulated lipid droplets in cells. Inset images: higher magnification views of the adipocyte-like
cells from the same culture shown in the main images. Scale bars: main images, 100 μm; insets, 50 μm.
(C, F, I) Neurogenic induction. βIII-tubulin: red fluorescence; 4',6-diamidino-2-phenylindole
dihydrochloride (DAPI): blue fluorescence. Scale bars: (C) 20 μm; (F, I) 30 μm. (J) Myogenic induction
and RT-polymerase chain reaction analysis of gene expression. M, RNA from cultured human myoblasts
(obtained from Dr. Wesley M. Jackson, NIH/NIAMS, Bethesda, MD); C, uninduced control; m,
induction in myogenic medium. SCAP, stem cells from apical papilla; DPSC, dental pulp stem cells;
BMMSCs, bone marrow mesenchymal stem cells.

Growth of stem cells in PLG

SCAP or DPSCs (5 × 105/50 μL) were seeded onto a PLG copolymer scaffold (75:25 molar
ratio) cut into ~1.5–2 mm3, cultured in vitro for 10 days or up to 8 weeks, and processed for
histological or SEM analysis. Cells seeded onto PLG attached well as revealed by SEM
analysis shown in Figure 3A, B, E, and F. The hemotoxylin and eosin staining showed that
blank PLG scaffolds were completely colorless (not shown), suggesting that the eosin
staining seen in the DPSC-seeded PLG represented the cytoplasm of cells and extracellular
matrix (Fig. 3C, D). SEM analysis of the long-term cultures (8 weeks) revealed that cells
laid down fiber-like matrix (Fig. 3E, F). Minimal or no contraction of cells/PLG constructs
was noted after 7–8 weeks in cultures.

FIG. 3.
Cell attachment and growth in the PLG scaffold in vitro. Scanning electronic
microscopy of DPSCs seeded onto PLG after (A) 10 days and (B)14 days in culture.
(C, D) H&E staining of DPSCs seeded onto PLG after 7 weeks in culture. (E, F)
Scanning (more ...)

FIG. 3.
Cell attachment and growth in the PLG scaffold in vitro. Scanning electronic microscopy of DPSCs
seeded onto PLG after (A) 10 days and (B)14 days in culture. (C, D) H&E staining of DPSCs seeded
onto PLG after 7 weeks in culture. (E, F) Scanning electronic microscopy of SCAP seeded onto PLG
after 8 weeks in culture. Scale bars: (A) 200 μm; (B, D, E) 20 μm; (C) 250 μm; and (F) 1 μm. Arrows in
(A) indicate attached cells, in (B) scaffold surface, in (E and F) fiber-like matrix. PLG, poly-D,L-lactide
and glycolide; H&E, hemotoxylin and eosin. Color images available online at
www.liebertonline.com/ten.

In vivo pulp/dentin tissue regeneration

Ingrowth of subcutaneous tissue into emptied root canal space
The control root fragment did not receive any cell-seeded PLG in the emptied root canal
space. Three months after being transplanted into the mouse subcutaneous space, the canal
space appeared to be filled with the subcutaneous connective tissue, mainly adipose tissue,
which grew more than 2 mm into the canal (Fig. 4). The other half (to the MTA side) of the
canal space appeared empty, or the tissue was lost during the sample processing.

FIG. 4.
Histological analysis of control group in the mouse subcutaneous model. (A) A root
fragment with empty canal space was transplanted into a SCID mouse for 3 months
before being removed and processed for H&E staining. Mouse subcutaneous tissue
ingrown (more ...)

FIG. 4.
Histological analysis of control group in the mouse subcutaneous model. (A) A root fragment with
empty canal space was transplanted into a SCID mouse for 3 months before being removed and
processed for H&E staining. Mouse subcutaneous tissue ingrown from the canal opening (arrow). ( B)
Higher magnification view showing the ingrown fatty tissue (arrow). Scale bars: (A) 1 mm; (B) 0.5 mm.

Vascularized pulp tissue regeneration in emptied root canal space

Longitudinal sections of the root constructs inserted with PLG seeded with SCAP or
DPSCs revealed that the emptied canal was filled with regenerated pulp-like tissue as
shown in Figures 5 and and6.6. Voids were scattered in the pulp-like tissue, which was
most likely the result of unresorbed PLG scaffolds. There was a sharp distinction between
the histological characteristics of the regenerated pulp-like tissue situated only within the
canal space and those of the subcutaneous soft tissues in the mouse dermal space. A thin
layer of fibrous capsule separated the two types of tissues. The entire pulp-like tissue was
vascularized with a uniform cell density resembling the natural pulp (Fig. 5I).

FIG. 5.
Histological analysis of in vivo pulp/dentin regeneration using SCAP. A root
 fragment was prepared and the canal space inserted with SCAP/PLG and
transplanted into a SCID mouse for 3 months. The sample was harvested and
processed for H&E staining. (more ...)

FIG. 5.
Histological analysis of in vivo pulp/dentin regeneration using SCAP. A root fragment was prepared and
the canal space inserted with SCAP/PLG and transplanted into a SCID mouse for 3 months. The sample
was harvested and processed for H&E staining. D, original dentin; rD, regenerated dentin-like tissue; rP,
regenerated pulp-like tissue. Blue arrow in (A) indicates the blood supply entrance; green arrows in (B)
and (C) indicate continuous layer of uniformed thickness of rD; yellow arrows in (E) and (F) indicate
the region of well-aligned odontoblast-like cells with polarized cell bodies; green arrows in (G and H)
indicate junctions between D and rD. Scale bars: (A) 1 mm; (B and C) 500 μm; (D) 100 μm; (E and F)
20 μm; and (G–I) 50 μm.

FIG. 6.
Histological analysis of in vivo pulp/dentin regeneration using DPSCs. Samples were
prepared using the same procedures as described in Figure 5, except that the sample
was harvested from the SCID mouse 4 months after implantation. D, original dentin;
(more ...)

FIG. 6.
Histological analysis of in vivo pulp/dentin regeneration using DPSCs. Samples were prepared using the
same procedures as described in Figure 5, except that the sample was harvested from the SCID mouse 4
months after implantation. D, original dentin; rD, regenerated dentin-like tissue; rP, regenerated pulp-
like tissue. Od, odontoblast-like cells. Green arrows in (A) indicate rD; blue arrows in (A) indicate the
entrance of blood supply; blue arrows in (B and C) indicate the thin layer of rD under MTA cement;
blue arrows in (F and G) indicate the junction of D and rD; black arrow in (G) indicates well-aligned
odontoblast-like cells. Yellow arrows in (G) indicate dentinal tubule-like structures. Scale bars: (A) 1
mm; (B) 200 μm; (C–E) 100 μm; and (F and G) 50 μm. Color images available online at
www.liebertonline.com/ten.

Formation of dentin-like tissue and odontoblast-like cells.

A representative sample using SCAP/PLG showed a continuous layer of mineralized tissue
with uniform thickness (~200 μm) deposited onto the existing dentinal walls and onto the
MTA cement surface in the canal (Fig. 5); herein referred to as “regenerated dentin-like”
tissue. When using DPSCs/PLG, a layer of regenerated dentin-like tissue was also
deposited onto the canal dentinal walls (Fig. 6). In this sample, the regenerated dentin-like
tissue had less continuity and thickness compared with the sample shown in Figure 5.
Higher magnification observations revealed that a layer of odontoblast-like cells were lined
against the mineralized dentin-like tissue (Figs. 5 and and6).6). There were also scattered
cells embedded within the dentin-like structure. Some located between the original dentin
and the regenerated dentin. The newly deposited dentin-like tissue appeared to tightly
adhere to the original dentin except where there were gaps that resulted from sample
processing artifacts. The newly formed mineralized tissue also appeared to fill into the
space of dentinal tubules (Fig. 5H). At higher magnification, unlike the natural dentin, the
pulp side of the regenerated dentin-like tissue was not smooth; instead, there were
projections of the structures into the pulp side (Figs. 5 and and66).
The odontoblast-like cells were not well organized, nor well aligned as the natural
counterparts, and it was difficult to observe the typical characteristics that natural
odontoblasts possess (e.g., polarized cell bodies). Nonetheless, some regions of the
odontoblast-like cells showed somewhat typical odontoblast characteristics. Groups of
odontoblast-like cells were well aligned, such that each cell was spatially juxtaposed to
adjacent cells with overt polarized morphology (Fig. 5E, F, and Fig. 6G). The regenerated
dentin-like tissue did not form observable well-organized dentinal tubules, except in a few
regions where odontoblast-like cells were better aligned (Fig. 6G).
Cells in regenerated pulp-like tissue are of human origin.
To verify that the cells responsible for generating the pulp/dentin-like tissues in the canal
space were of human origin, antibodies against human mitochondria were used to detect
human cells in the tissues. As shown in Figure 7D–F, a majority of the cells stained
positively for the antibodies, confirming the human cell origin of these cells in the
regenerated pulp-like tissue.

FIG. 7.
Immunohistochemical analysis of pulp tissue regenerated in vivo using SCAP. (A–C)
H&E staining; immunostaining for (D–F) human mitochondria; (G) dentin
 sialoprotein; (H) alkaline phosphatase and (I) bone sialoprotein. D, original (more ...)

FIG. 7.
Immunohistochemical analysis of pulp tissue regenerated in vivo using SCAP. (A–C) H&E staining;
immunostaining for (D–F) human mitochondria; (G) dentin sialoprotein; (H) alkaline phosphatase and
(I) bone sialoprotein. D, original dentin; rD, regenerated dentin-like tissue; rP, regenerated pulp-like
tissue. Arrows indicate cells with positive staining. Color images available online at
www.liebertonline.com/ten.

Odontoblast-like cells express odontogenic markers

To further verify the dentin-like tissue was generated by cells of odontoblast lineage,
several odonto/osteogenic markers were examined immunohistochemically. Positive
staining for DSP, BSP, and ALP was seen in cells lining against as well as those embedded
in the newly deposited dentin-like tissues, suggesting that the differentiated odontoblast-
like cells were responsible for the production of the calcified tissue (Fig. 7G–I).
In addition, we compared CD105 expression in the regenerated and the natural pulp.
CD105 was expressed by DPSCs and SCAP based on the flow cytometry analysis
presented in Table 1. Immunocytochemical analysis of SCAP in cultures confirmed their
CD105 expression (Fig. 8A). In the natural pulp, CD105 expression was detected in
association with blood vessels and in odontoblasts (Fig. 8C, D). CD105 (Endoglin) is
known to be expressed on human vascular endothelial cells. In comparison, CD105 was
also expressed by odontoblast-like cells and paravascular cells in the regenerated pulp-like
tissus (Fig. 8E, F), suggesting that the regenerated tissue closely resembles natural pulp.

FIG. 8.
Immunocyto/histochemical staining of CD105. Cultured SCAP immunostained with
(A) anti-CD105 antibody or (B) nonimmune IgG as negative control. (C) Positive
staining of odontoblasts (arrowheads) in a sample of natural human dental
 pulp/dentin tissue. ( (more ...)

FIG. 8.
Immunocyto/histochemical staining of CD105. Cultured SCAP immunostained with (A) anti-CD105
antibody or (B) nonimmune IgG as negative control. (C) Positive staining of odontoblasts (arrowheads)
in a sample of natural human dental pulp/dentin tissue. (D) A blood vessel in the natural pulp is positive
for CD105 (arrow). (E) Positive immunostaining is seen in odontoblast-like cells lining against rD or
inside rD (arrowheads). (F) CD105-positive cells are seen in rP and around a blood vessel (arrow). rD,
regenerated dentin-like tissue; rP, regenerated pulp-like tissue. Color images available online at
www.liebertonline.com/ten.

  Other Sections▼
o Abstract
o Introduction
o Materials and Methods
o Results
o Discussion
o References

Discussion
Our study is the first in vivo evidence demonstrating de novo synthesis of vascularized
human pulp/dentin-like tissues in an emptied human root canal space 5–6 mm deep with an
opening end of only ~2.5 mm. The most striking phenomenon is the formation of a
continuous layer of dentin-like tissue on the existing canal dentinal walls and on MTA
cement surface. Expression analysis of several key genes, including DSP, BSP, ALP, and
CD105, indicates that the regenerated pulp tissues closely resemble natural pulp tissue. Our
findings constitute the first step toward stem cell–mediated de novo regeneration of
pulp/dentin in clinical endodontic practice. Use of heterogeneous population of
stem/progenitor cells appeared to be able to achieve this purpose. Both SCAP and DPSCs
as mesenchymal-like stem cells presented a different profile of multipotency from
BMMSCs. This may be because of their different developmental origin—derived from or
associated with neural crest cells.
Previously, we discussed several issues regarding the establishment of a protocol for
pulp/dentin regeneration, including the scaffolds suitable for this purpose, the extent of
vascularization of the regenerated pulp, and the formation of odontoblasts and new dentin
on the existing dentin surface.4,5,10,16 Our previous in vitro studies showed that although
collagen gels are a convenient cell carrier for pulp regeneration in the root canal space,
severe contraction may hamper the regeneration process. 10 A recent in vivo study targeting
root dentin perforation repair with the use of DPSCs and dentin matrix protein 1 carried by
collagen matrix showed that soft connective pulp-like tissue was formed in the perforated
site, but no hard tissue was generated.18 Regeneration of a small portion of pulp after
pulpotomy using a collagen matrix appears to be successful as demonstrated in a dog study
model.19 Biosynthetic scaffolds such as PLG or poly-L-lactic acid are much more resistant
to contraction than collagen. They are generally made into porous form to provide inner
spaces for cells to populate. Cordeiro et al. reported successful regeneration of well-
vascularized pulp-like soft tissue utilizing poly-L-lactic acid as a scaffold fabricated in the
pulp chamber space of a tooth slice that was seeded with stem cells from human exfoliated
deciduous teeth.6 Together with our findings in this study, synthetic polymers of lactide
and/or glacolide appeared to be suitable scaffold systems for de novo pulp regeneration. We
speculated that the slow degradation of the PLG in our study negatively affected the quality
of the regenerated pulp-like tissue. In future studies, modification of the ratio of lactide and
glycolide or selection of other type of scaffold materials such as silk may improve this
situation.20,21
Vascularization in the regenerated pulp tissue in canal space was a major concern because
the blood supply for the space can only come from the apex.4 The above mentioned studies
have tried to avoid this issue in testing pulp regeneration by using a thin tooth slice
model.6,18 Utilizing factors such as vascular endothelial growth factor and/or platelet-
derived growth factor to enhance angiogenesis or the addition of vascular cells or
hematopoietic progenitors has been an effective approach to resolve to this issue for tissue
regeneration.14,22–25 In this study, angiogenic enhancement was not employed. The tooth
fragments contained a canal space 5–6 mm deep; therefore, it was likely that the blood
vessel growth into the end of the canal space to provide nutrients for the stem cells could
not occur shortly after the tooth fragment was transplanted into the mouse subcutaneous
space. However, our results indicate that cells not only survived well but also were able to
regenerate tissues. It is possible that the nutrients were able to diffuse into the canal space,
allowing the stem cells to build new tissues; once the blood vessels were generated in the
entire canal space, long-term stability of the tissue was established. The question arises as
to whether a smaller opening of the canal, that is, <2 mm, will affect the efficacy of tissue
regeneration in the coronal end of the canal. Further studies are needed to test the limit of
the canal opening size that allows pulp regeneration and whether the addition of angiogenic
factors can compensate the smaller canal opening.
It was unknown whether de novo regenerated pulp in the canal space can produce new
dentin onto the existing dentin. To generate new dentin, stem cells must first differentiate
into odontoblasts. It was not clear whether using a scaffold such as PLG, without osteo-
inductive properties, will lead to stem-cell differentiation into odontoblasts. However,
regenerating pulp/dentin in the root canal space is not the same as regenerating bone. Pulp
and dentin in the canal space have their specific locations; therefore, any scaffold system
that is osteoinductive, such as hydroxyapatite and tricalcium phosphate, is in fact not
appropriate for pulp/dentin in the root canel space regeneration. This approach would likely
generate scattered calcified tissue in the entire canal space.
In vivo studies have demonstrated that stem cells from human exfoliated deciduous teeth
were able to differentiate into odontoblast-like cells lining against the existing dentin
surface.6 These findings suggest that existing dentin is sufficient to guide stem cells in the
canal space to differentiate into odontoblast-like cells. Even the chemical treatment of
dentin did not appear to affect this capacity. From these observations, it appears that stem
cells seeded in the scaffold are attracted to the dentinal wall, differentiate into odontoblast-
like cells, and extend their cellular processes into the dentinal tubules. The mechanism
underlying this phenomenon has been speculated to be the release of growth factors such as
transforming growth factor-β that is known to be embedded in dentin, 26,27 which attracts and
induces odontoblast differentiation of the seeded stem cells. 10 Chemical disinfection of the
root canal space may damage these embedded growth factors. Further investigation is
needed to seek ways to avoid this potential damage.
The most interesting and important finding of this study is the formation of a continuous
layer of dentin-like tissue with uniform thickness on the existing canal dentin walls and the
MTA cement surface, especially with SCAP-seeded samples. This fulfills, at least in part, a
major requirement of functional tissue engineering/regeneration because dentin production
is one of the major functions of pulp. However, the characteristics of the regenerated
dentin-like tissue are quite different from the natural dentin. First, dentinal tubules are not
obvious, possibly because of their convoluted paths. Second, it is quite cellular, and third,
the formation is not synchronous. There were only some regions of the new dentin-like
tissue that showed dentinal tubule-like structures (Fig. 6G). Our previous in vitro work
demonstrated that DPSCs seeded onto a processed dentin surface morphologically
transformed into odontoblast-like cells each with a cellular process extending into the
dentinal tubule.10,15 Based on this observation along with the present in vivo findings, the
following events were likely to have occurred subsequent to in vivo transplantation of the
tooth fragments: (i) some SCAP or DPSCs on PLG in the canal space migrate toward the
dentin surface, (ii) these cells receive signals from dentin and differentiate into odontoblast-
like cells, (iii) a cellular process is extended from each cell into the dentinal tubules, (iv)
cells initiate production of extracellular matrix in the dentinal tubule space as well as onto
the dentin surface, and (v) the remaining steps of dentin-like tissue apposition are similar to
the natural dentin production, except cells laid down dentin-like tissue at a different pace.
The question remains as to whether every dentinal tubule is occupied by a newly
differentiated odontoblast-like cell of equal potentiality. If not, it may explain the
disorganized nature of the newly synthesized dentin-like tissue and the alignment of those
odnotoblast-like cells as well as the entrapment of many of these cells in the dentin-like
structure. Tertiary dentin produced by the natural pulp has less organization of dentinal
tubules and may have cell entrapment in the dentin; therefore, the newly regenerated
dentin-like tissue may be similar to tertiary dentin.
Taken together, our findings show that pulp/dentin regeneration can be accomplished with
a stem/progenitor cell–mediated tissue engineering approach. This study is the first step
toward reaching the goal of regenerating functional pulp/dentin that is very similar to its
natural counterparts. To achieve this goal, future investigations must include the use of
larger animals, such as minipigs, to regenerate these tissues in situ in the jaw bone. Besides
gaining vascularity, the innervation of the regenerated pulp/dentin tissue by ingrowth of
nerve fibers from the apical tissues requires extensive investigation. Further, if a higher
 quality of the regenerated dentin tissue is desired, that is, producing new dentin with well-
organized dentinal tubules, the method of treating dentin surface may need modification to
provide better signaling stimulation to guide stem cell differentiation toward odontoblast
lineage in a more synchronous manner.

Acknowledgments
This work was supported in part by grants from the National Institutes of Health (NIH) R01
DE019156-01 (G.T.-J.H.), RO1 DE17449 (S.S.), by the NIAMS/NIH Intramural Research
Program Z01 AR41131 (R.S.T), and by an Endodontic Research Grant from American
Association of Endodontists Foundation (G.T.-J.H.). We wish to thank Dr. Matthew
Daniels (NIH/NHLBI, Electron Microscopy Core) for the assistance of SEM analysis, Dr.
Larry Fisher (NIH/NIDCR) for providing antibodies, Dr. Wesley M. Jackson
(NIH/NIAMS) for providing the RNA from cultured human myoblasts, and Dr. Yi Liu
(USC) for the assistance of neurogenic studies.

Disclosure Statement
No competing financial interests exist.

  Other Sections▼
o Abstract
o Introduction
o Materials and Methods
o Results
o Discussion
o References

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